U.S. patent number 6,160,103 [Application Number 09/147,420] was granted by the patent office on 2000-12-12 for conjugates of an oligonucleotide/electronic conductor polymer with a molecule of interest, and their uses.
This patent grant is currently assigned to Cis Bio International. Invention is credited to Herve Bazin, Joseph Marchand.
United States Patent |
6,160,103 |
Marchand , et al. |
December 12, 2000 |
Conjugates of an oligonucleotide/electronic conductor polymer with
a molecule of interest, and their uses
Abstract
The invention concerns a macromolecule of formula (I): P-O-M in
which M represents a molecule of interest, O represents an
oligonucleotide chain; P represents a monomer of an electronic
conductor polymer, and x and y are integers equal to 1 or more. The
invention also concerns the copolymers obtained from the P-O-M
macromolecules.
Inventors: |
Marchand; Joseph (Orsay,
FR), Bazin; Herve (Impasse Laennec, FR) |
Assignee: |
Cis Bio International (Saclay,
FR)
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Family
ID: |
9493371 |
Appl.
No.: |
09/147,420 |
Filed: |
December 21, 1998 |
PCT
Filed: |
June 25, 1997 |
PCT No.: |
PCT/FR97/01134 |
371
Date: |
December 21, 1998 |
102(e)
Date: |
December 21, 1998 |
PCT
Pub. No.: |
WO97/49718 |
PCT
Pub. Date: |
December 31, 1997 |
Foreign Application Priority Data
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Jun 25, 1996 [FR] |
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96 07846 |
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Current U.S.
Class: |
536/23.1;
435/287.2; 435/5; 435/6.11; 530/402; 536/22.1; 536/24.1 |
Current CPC
Class: |
C07H
21/00 (20130101); C08G 61/124 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C08G 61/00 (20060101); C08G
61/12 (20060101); C07H 021/02 (); C07H 019/00 ();
C07K 001/00 (); C07K 014/00 (); C07K 016/00 () |
Field of
Search: |
;435/5,6
;536/22.1,23.1,24.1,25.3 ;530/402 |
Foreign Patent Documents
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WO94/22889 |
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Oct 1994 |
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WO |
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WO95/29199 |
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Nov 1995 |
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WO |
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Other References
Solomons "Organic Chemistry, Fifth Edition" John Wiley and sons,
Inc. pp. 997-1000, 1992.
|
Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt, P.C.
Claims
What is claimed is:
1. A macromolecule of the formula (I):
in which:
M represents a molecule of interest selected from the group
consisting of a protein, a peptide, an amino acid, a glycopeptide,
a lipid, a steroid, a glycolipid, a sugar and a polysaccharide;
O represents an oligonucleotide chain;
P represents a monomer of an electronically conductive polymer.
2. The macromolecule according to claim 1, wherein the monomer P
represents a pyrrole group.
3. The macromolecule according to claim 1, wherein the
oligonucleotide chain O consists of a single-stranded
oligonucleotide.
4. The macromolecule according to claim 1, wherein the
oligonucleotide chain O consists of an oligonucleotide which is
double-stranded along at least a part of its length.
5. The macromolecule according to claim 1, wherein the
oligonucleotide chain O comprises at least 6 nucleotides and in
that its percentage of (G+C) is less than or equal to 70%.
6. A process for preparing a macromolecule P-O-M of the formula
(I):
in which:
M represents a molecule of interest;
O represents an oligonucleotide chain;
P represents a monomer of an electronically conductive polymer,
from a mixture which comprises the said macromolecule and also the
P-O and M reagents from which it was formed, which process
comprises at least one step during which the said mixture is
fractionated by any means which make it possible to separate the
fractions comprising M and P-O-M, respectively, from the fraction
comprising P-O, and a step during which the fraction comprising
P-O-M is detected and/or the quantity of P-O-M is determined by
detecting and/or measuring a parameter which is associated with the
oligonucleotide O and not associated with the molecule of interest
M.
7. The process according to claim 6, wherein the said parameter
which is associated with the oligonucleotide O and not associated
with the molecule of interest M is absorption in the ultraviolet at
a wavelength of between 240 and 270 nm.
8. A copolymer, wherein at least one of its units consists of a
macromolecule according to claim 1.
9. An electrode, wherein its surface carries at least one copolymer
according to claim 8.
10. An electrode matrix, which comprises at least one electrode
whose surface carries at least one copolymer, wherein at least one
of the units of said copolymer consists of a macromolecule
according to claim 1.
11. The electrode matrix according to claim 10, which comprises at
least two electrodes which differ from each other in at least one
of the monomers P, the oligonucleotides O and/or the molecules M
which are present on their surface.
12. The electrode matrix according to claim 10, which comprises at
least two electrodes which differ from each other in the quantity
of oligonucleotides O and/or molecules M per unit of surface
area.
13. The electrode matrix according to claim 10, which additionally
comprises at least one electrode which is covered with an
ECP/oligonucleotide copolymer.
14. A method of monitoring the attachment of at least one molecule
X to an electrode, comprising:
electropolymerizing a copolymer on a surface of an electrode
wherein at least one of the monomer units of said copolymer
consists of a macromolecule of the formula (I):
in which:
M represents a molecule of interest;
O represents an oligonucleotide chain;
P represents a monomer of an electronically conductive polymer,
contacting said electrode having said copolymer on the surface with
said molecule X,
wherein said molecule X comprises at least one of the constituents
P, O or M of a macromolecule of formula (I) and said monitoring is
qualitative and/or quantitative.
15. The method of claim 14, wherein said copolymer and said
molecule X are on the same electrode.
16. The method of claim 14, wherein said copolymer and said
molecule X are on two different electrodes.
17. The method of claim 16, wherein the two different electrodes
belong to two different matrices.
Description
The present invention relates to novel methods and novel compounds
for controlling the attachment of different molecules of interest
to an electronically conductive polymer (ECP);
The application PCT WO 94/22889 in the name of CIS BIO
INTERNATIONAL (inventors, TEOULE et al.) describes the attachment
of oligonucleotides to a support which consists of an
electronically conductive polymer (ECP).
This attachment can be effected in a variety of ways:
1) by the attachment of a presynthesized oligonucleotide to the ECP
(that is by chemically reacting the oligonucleotide on the
previously functionalized ECP or by copolymerizing ECP monomers
with the product of the condensation of the oligonucleotide on one
of the said monomers).
2) by elongation of the oligonucleotide starting with one
nucleoside, one nucleotide or one oligonucleotide which has already
been attached to the ECP using, for example, one of the standard
methods for synthesizing nucleic acids.
The attachment of oligonucleotides to ECPs facilitates the
isolation and use of oligonucleotide matrices which can, in
particular, be employed for sequencing nucleic acids and for
diagnosis.
Just like oligonucleotide matrices, peptide matrices and, more
generally, matrices of various molecules represent a particularly
advantageous tool, for example in the field of diagnosis or for
screening active molecules. It would therefore be desirable for
other types of matrix to be able to benefit from the improvements
brought about by using ECPs.
However, the attachment of molecules of interest other than
oligonucleotides, for example the attachment of peptides, to an ECP
poses more problems than does the attachment of
oligonucleotides.
Thus, although methods for synthesizing pyrrole carrying an amino
acid or a dipeptide are described in the literature [GARNIER et al.
J. Am. Chem. Soc., 116, 8813-8814 (1994)], these are methods for
carrying out synthesis in solution, which methods do not in
practice enable synthetic peptide molecules of a size greater than
2 or 3 amino acids to be obtained in sufficient yield. However,
molecules which are of real interest within the field of diagnosis
or that of screening active molecules are longer; for example, a
"minimal" antigenic motif generally contains an average of 6 amino
acids.
The usual methods of synthesizing peptides on a solid support,
which methods are derived from the MERRIFIELD technique, involve
the use of different groups for protecting the side chains of the
amino acids. The elimination of these groups, and the separation of
the peptide and support at the conclusion of the synthesis are
effected in strong acid medium (hydrofluoric acid or
trifluoroacetic acid). However, ECP monomers, in particular the
pyrrole residue, have a tendency to polymerize in acid medium,
thereby creating undesirable by-products.
It seems, therefore, that neither the synthesis method which is
carried out in solution, and whose possibilities are limited to
synthesizing dipeptides or tripeptides, nor synthesis on a support,
whose implementation requires chemical conditions which are
incompatible with the stability of the pyrrole residue, are
suitable for synthesizing peptides which are modified by a pyrrole
residue.
It is nevertheless possible to graft an ECP monomer, for example a
pyrrole residue, onto a preformed oligopeptide which has been
obtained by traditional peptide synthesis on a solid phase. This
grafting can be effected using known methods, for example by
reaction between a carboxylic derivative of the pyrrole (activated
by a coupling reagent) and one of the available amino functions of
a peptide (for example the amino function at the N-terminal end),
in accordance with the following scheme [S. E. WOLOWACZ et al.,
Anal. Chem., 64, 1541-1545, (1992)].
Pyrrole--COOR+NH2--peptide.fwdarw.Pyrrole--CONH--peptide
At the conclusion of the reaction, the pyrrole-peptide conjugate
has to be isolated from the reaction mixture containing the
unreacted peptide and pyrrole as well as any possible salts and
by-products.
The various methods which it might therefore be possible, a priori,
to envisage using for this purpose are those which are customarily
employed for purifying peptides, in particular reverse-phase
chromatography methods (RP-HPLC), or gel filtration.
The simplest way to detect the peptides at the conclusion of the
chromatography is to measure the ultraviolet absorption at a
wavelength of from 215 nm to 220 nm; this is because all peptides
absorb light at this wavelength. However, this approach of
measuring absorption in the region of 215 nm suffers from the
drawback of being relatively insensitive and of not being specific
since solvents and organic or inorganic ions are also detected.
Methods exist which enable the peptides to be detected specifically
by an "in-line" chemical reaction at the outlet of the
chromatography column. While this method has the advantage of
increasing the sensitivity of the detection, it makes this
detection more cumbersome and, furthermore, irreversibly modifies
the peptide; this modification can, in particular, have significant
consequences for its functional properties (for example its
antigenicity).
The problems expounded above with regard to peptides also arise
when detecting other molecules of interest, etc. Thus, a large
number of substances, such as sugars, polysaccharides or steroids,
either do not exhibit any specific absorption at a defined
wavelength in the UV or only exhibit weak absorption which in turn
prejudices the sensitivity of the detection.
One is therefore obliged to use an appropriate physical or chemical
means to detect the presence of the sought-after molecule in each
fraction derived from the chromatography, something which, quite
obviously, takes a large amount of time; furthermore, this analysis
often destroys the analyte in question.
The object of the present invention is that of obtaining
conjugates, which are easy to synthesize and purify, and molecules
of interest with an ECP monomer. With this aim, the inventors have
prepared conjugates which possess properties which are not
naturally possessed either by the molecule of interest or by the
ECP monomer.
In these conjugates, the molecule of interest and the ECP monomer
are linked by way of an oligonucleotide chain which serves as a
spacer arm between the ECP monomer and the molecule of interest
under consideration.
The present invention relates to a macromolecule of the following
formula (I):
in which:
M represents a molecule of interest;
O represents an oligonucleotide chain;
P represents a monomer of an electronically conductive polymer.
Within the meaning of the present invention, a "molecule of
interest" is understood as being any molecule which exhibits a
useful functionality in reactions carried out on a solid support,
for example synthesis reactions or direct or indirect detection
reactions. This molecule of interest can, for example, without this
list being limiting, be a biomolecule, such as a protein (in
particular an enzyme), an amino acid, a peptide, a glycopeptide, a
lipid, a steroid, a glycolipid, a sugar, a polysaccharide, a
molecule which is able, directly or indirectly, to generate a
signal, or else a complex, multifunctional molecule, etc.
Advantageously, this molecule of interest is one of the members of
an affinity couple and can, for example, be biotin or a potentially
antigenic peptide, etc.
P can, for example, be a monomer of polyacetylene, of polyazine, of
poly(p-phenylene), of poly(p-phenylene vinylene), of polypyrene, of
polypyrrole, of polythiophene, of polyfuran, of polyselenophene, of
polypyridazine, of polycarbazole, of polyaniline, etc.
Advantageously, P is a pyrrole group.
The oligonucleotide chain O can consist of an assembly of natural
nucleotides and/or nucleotide analogues, such as those described,
for example, by UHLMANN, [Chemical Review, 90:4, 543-584 (1990)].
It can be a single-stranded oligonucleotide or a double-stranded
oligonucleotide over at least a part of its length. In the second
case, one of the strands is covalently attached to the P monomer
and the other strand is covalently attached to the molecule of
interest M.
In theory, there is no limit to the nature or length of the
oligonucleotide; in practice, the oligonucleotide chain
advantageously has a length of between 6 and 60, preferably between
10 and 30, nucleotides. Advantageously, the percentage of (G+C) in
the oligonucleotide O is less than or equal to 70%, preferably less
than or equal to 50%.
The attachment of the oligonucleotide O to the ECP monomer can be
effected as described in application PCT WO 94/22889. The
attachment of the molecule of interest M to the oligonucleotide O
can be effected by means of various methods, which are known per
se, for linking an oligonucleotide to another molecule. The choice
of the most appropriate method essentially depends on the nature of
the molecule of interest M.
For example, the oligonucleotide O can be activated by attaching an
ester of N-hydroxysuccinimide, an amino acid, an SH group [ARAR et
al., Bioconjugate Chem. 6, p. 573-577, (1995)] or a maleimide
group.
The present invention also relates to a process for preparing a
macromolecule P-O-M as defined above from a mixture which comprises
the said macromolecule and also the P-O and M reagents from which
it was formed, which process is characterized in that it comprises
at least one step during which the said mixture is fractionated by
any means which make it possible to separate the fractions
comprising M and P-O-M, respectively, from the fraction comprising
P-O, and a step during which the fraction comprising P-O-M is
detected and/or the quantity of P-O-M is determined by detecting
and/or measuring a parameter which is associated with the
oligonucleotide O and not associated with the molecule of interest
M.
By its chemical nature and its length, the oligonucleotide chain O
plays a role in the physical properties (polarity in
chromatography, for example) of the conjugate with the molecule of
interest under consideration, a fact which makes it possible to
arrange optimum separation of the resulting conjugate from the
excess reagents. Moreover, the oligonucleotide chain enables the
conjugate to be detected specifically at a wavelength of between
240 and 270 nm (advantageously 265 nm) in the ultraviolet and,
where appropriate, by means of hybridization with an
oligonucleotide having a complementary sequence.
The properties conferred by the oligonucleotide chain O thus permit
easy manipulation of minute quantities of molecules of interest by
facilitating their separation using ultrafiltration and/or
partition chromatography methods or methods of affinity
chromatography on oligonucleotides having a complementary sequence,
and their detection and rapid quantification by means of measuring
specific absorption between 240 and 270 nm. This is particularly
advantageous when the molecule of interest M is a peptide; this is
because measuring the absorption of the oligonucleotides at 265 nm
is much more sensitive and specific (there is no interference from
salts and solvents) than measuring the absorption of the peptides
at 205 nm.
The present invention also relates to copolymers at least one of
the units of which consists of a macromolecule P-O-M as defined
above.
Polymers in accordance with the invention are, for example, defined
by one of the following formulae (II), (III) or (IV): ##STR1## in
which: x and y represent integers equal to or greater than 1,
and
M represents a molecule of interest;
O represents an oligonucleotide chain
P represents a monomer of an electronically conductive polymer.
In a copolymer according to the invention, the electronically
conductive polymer monomers P may be identical to each other or
else differ from each other; the oligonucleotides O may also be
identical to each other or differ in their sequence and/or their
size and/or their single-stranded or double-stranded nature;
similarly, the M molecules may be identical or different from each
other.
These copolymers may be prepared by copolymerizing one or more
purified P-O-M conjugate(s), or such as defined above, with P
monomers and/or with P-O conjugates, such as those described in
application PCT WO 94/22889, and/or with P-M conjugates; they may
also be obtained by attaching one or more molecules of interest M
to all or some of the oligonucleotide side chains of an
ECP/oligonucleotide copolymer such as those described in
application PCT WO 94/22889. This attachment may, for example, be
effected by covalently linking a molecule of interest M to an
oligonucleotide O which is itself attached to the ECP, or else by
way of an oligonucleotide which is hybridized to the said
oligonucleotide O along at least some of its length.
The copolymers according to the invention may be used in all the
applications in which it is customary to attach molecules of
interest to a solid support, in particular to an electrode.
The present invention relates to electrodes whose surface carries
at least one copolymer in accordance with the invention.
For example, the surface of an electrode in accordance with the
invention may be entirely covered with one single copolymer in
accordance with the invention; it may also carry several copolymers
which are in accordance with the invention and which differ from
each other by at least one of the constituents P, O or M; this or
these copolymers may also be associated, on the same electrode,
with other polymers, for example ECP/oligonucleotide copolymers
such as those described in application PCT WO 94/22889 or else with
polymers of a different nature, such as, for example, ECPs which
result from polymerizing P monomers such as defined above.
Polymers in accordance with the invention can be used for building
matrices of molecules of interest, in particular electrode matrices
which comprise at least one electrode in accordance with the
invention, such as defined above.
Electrodes which constitute different points of one and the same
matrix may differ from each other in the P monomers which are
included in the composition of the copolymers which are present on
their surface and/or in the O oligonucleotide and/or the M
molecules of the side chains of these copolymers; they may also
differ from each other in the quantity of these side chains per
unit of surface area.
Some of the electrodes of one and the same matrix may carry a
polymer other than a copolymer in accordance with the invention,
for example an ECP/oligonucleotide copolymer such as those
described in application PCT WO 94/22889. Depending on the
envisaged use, this ECP/oligonucleotide copolymer may be retained
as such or else used for attaching other molecules of interest,
either directly or by means of hybridization with another
oligonucleotide which carries the molecule of interest.
Such matrices may be prepared by depositing desired polymers in a
targeted manner on pre-defined electrodes; for example, copolymers
in accordance with the invention may be deposited by the targeted
electrochemical copolymerization, on selected electrodes, of P-O-M
conjugates and/or of variable quantities of one and the same P-O-M
conjugate with P monomers and, where appropriate, P-O and P-M
conjugates. The use of macromolecules P-O-M and copolymers in
accordance with the invention therefore makes it possible to obtain
multifunctional matrices in a simple manner.
The P-O-M conjugates in accordance with the invention may also be
used for calibrating oligonucleotide matrices which are attached to
an electronically conductive polymer support. Thus, the inventors
observed that it was possible to establish a reproducible
correlation between the quantity of O-M side chains and the
quantity of oligonucleotides attached to an electronically
conductive polymer support. Consequently, measuring the attachment
of O-M side chains to an electrode under given experimental
conditions makes it possible to anticipate the quantity of
oligonucleotides which will be attached to another electrode (of
the same matrix or of another matrix) under the same experimental
conditions.
The use of P-O-M conjugates and copolymers in accordance with the
invention makes it possible to obtain electrodes which constitute
internal controls, making it possible qualitatively and/or
quantitatively to monitor the attachment of molecules X to a solid
support which consists of the surface of an electrode which carries
an electronically conductive polymer. This monitoring of the
attachment of molecules X can be effected at any time (after these
molecules have been deposited or while these molecules are being
used). The molecules X whose attachment can be detected by using a
copolymer in accordance with the invention may be of a variety of
natures; advantageously, they comprise at least one oligonucleotide
chain O and/or one molecule of interest M which can be identical
to, or different from, those of the copolymer in accordance with
the invention which is employed.
A polymer in accordance with the invention can be employed for
detecting and/or quantifying a molecule X which constitutes one of
the side chains of the same polymer, or else of a polymer of the
same formula, which has been deposited on the same electrode or
else on another electrode of the same matrix or of a different
matrix. It can also be employed for detecting and/or quantifying a
molecule X which belongs to a different polymer, which may also be
deposited on the same electrode or on another electrode of the same
matrix or of a different matrix.
This detection and/or quantification can be effected, for example,
by detecting oligonucleotide chains O of the copolymers in
accordance with the invention by means of hybridization with a
labelled probe or else by using, on one or more of the electrodes
of the matrix, copolymers in accordance with the invention which
comprise a molecule of interest M which constitutes a readily
detectable label (for example biotin or a fluorescent label).
The present invention will be better understood with the aid of the
remainder of the description which follows and which refers to
examples of preparing and using macromolecules and copolymers in
accordance with the invention.
EXAMPLE 1
Spectral and Chromatographic Properties of Peptides
A commercially available synthetic peptide (Cat. No. A2532
SIGMA-ALDRICH Chimie), which has a molecular mass of 1652.1 daltons
and which corresponds to ACTH (adrenocorticotrophic hormone)
fragment 11-24, is used to illustrate the problems posed by
detecting a peptide of biological interest. According to the
manufacturer's specifications, the peptide content of the
preparation is 61%.
25 .mu.l of a 2 mg/ml solution of the said peptide are injected
onto LICHROSPHER.RTM. RP-18E 5 .mu.m 125-4 a HPLC column (MERCK,
DARMSTADT, Germany). The column is eluted (1 ml/min) in 35 minutes
with the mixture A+B, (A=5% ACN (acetonitrile), 25 mM TEAAc
(triethylammonium acetate); B=50% ACN, 25 mM TEAAc) using a
gradient of from 0% to 100% B.
A peak at a retention time Rt=2 minutes and a relatively broad peak
at Rt=10 minutes are observed. The fractions corresponding to these
two peaks are collected, concentrated and analysed.
Fraction F1 (Rt=2 minutes) exhibits an absorption at A.sub.220 nm
=1.033. On the other hand, A.sub.275 nm is of the same order as the
background noise (0.005). This fraction therefore contains salts
(non-specific A.sub.220 nm).
Fraction F2 (Rt=10 minutes) exhibits an absorption at A.sub.220 nm
=1.90 and at A.sub.275 nm =0.073. This fraction therefore contains
the peptide.
This illustrates the fact that UV detection in the region of 215 to
220 nm does not, by itself, enable the signal on the chromatogram
due to the peptide to be differentiated from the interfering
signals of other substances.
The commercial synthetic peptide (Cat. No. A 0673, SIGMA-ALDRICH
CHIMIE), which has a molecular mass of 2465.7 daltons and which
corresponds to ACTH fragment (18-39), was analysed in the same way
by means of RP-HPLC. According to the manufacturer, the peptide
content of the sample is 82%.
The HPLC analysis is carried out under the same conditions as above
and the fractions are detected in the UV at 215 nm.
One major peak having an Rt=18.45 minutes is observed; this peak is
narrower than that observed with ACTH fragment (11-24).
This experiment demonstrates that two different peptide fragments
exhibit peaks which are characterized by very different retention
times. Comparison of the HPLC profiles also shows that the peaks
may be broadened, as in the case of ACTH (11-24).
EXAMPLE 2
Synthesis of an Oligonucleotide Which is Modified Both with a
Pyrrole Residue and with an Aminoalkyl Arm
A modified oligonucleotide having the sequence:
5'HO-dC.sup.pyrrole -(T).sub.10 -p-dC.sup.aminohexyl -p-dTOH3'
is synthesized on a solid support (CPG (controlled-pore glass))
using the "phosphite-phosphoramidite" method described by BEAUCAGE
and LYER [Tetrahedron., 48, 2223-2311, (1992)].
The main steps of this synthesis are described below.
An aminoalkyl-phosphoramidite derived from
5'-O-(4,4'-dimethoxytrityl)-N-4-(6-aminohexyl)-2'-deoxycytidine
[ROGET et al. Nucleic Acids Res., 17, 7643-7650, (1989)] is
prepared.
This aminoalkyl-phosphoramidite is obtained in two steps: the
primary amine of the alkylamine arm carried by the nucleoside is
protected with a trifluoroacetyl group, after which the 3' hydroxyl
is phosphitylated using a method which is analogous to that
described by SPROAT et al. [Nucleic Acids Res. 15, 6181-6196,
(1987)], in the case of the phosphoramidite of
5'-trifluoroacetamido-2',5'-dideoxythymidine [lacuna] by SPROAT et
al. [Nucleic Acids Res. 15, 6181-6196, (1987)].
The aminoalkyl-phosphoramidites which are thus obtained are coupled
on a column of a DNA synthesizer by way of thymidines which have
been previously attached to this column.
Other aminoalkyl-phosphoramidites, such as those described by
AGRAWAL et al. [Nucleic Acids Res., 14, 6227, (1986)]; CONNOLLY,
[Nucleic Acids Res. 15, 3131, (1987)]; and BEAUCAGE and LYER
[Tetrahedron. 49, 1925-1963, (1993)] can also be used.
The phosphoramidite of the thymidine is then condensed with the
aminoalkyl-phosphoramidite which is attached to the column. This
step is repeated until a 10-mer oligonucleotide chain has been
obtained.
A phosphoramidite of
5'-O-(4,4'-dimethoxytrityl)-N-4-(6-aminohexyl)-2'-deoxycytidine, to
which a pyrrole residue has been grafted in accordance with the
protocol described in application PCT WO 94/22889 and by LIVACHE et
al., [Nucleic Acids Res. 22, 2915-2921, (1994)], is condensed onto
the 5' end of the resulting oligonucleotide.
The oligonucleotide is deprotected in concentrated ammonia
(55.degree. C. for 16 hours) and purified by HPLC on a
LICHROSPHER.RTM. RP-18E 250-10 (10 .mu.m) column (MERCK, DARMSTADT,
Germany) using a gradient of acetonitrile in 50 mM triethylammonium
acetate (buffer A; 5% acetonitrile, buffer B: 50% acetonitrile;
flow rate 5 ml/minute, gradient of 10% B to 25% B in 20 minutes),
in accordance with the method described in: "Oligonucleotide
synthesis: A practical approach, Ed M. J. Gait. IRL Press,
Oxford".
The fractions corresponding to a major peak (retention time greater
than 15 minutes) are evaporated. After evaporation, the
oligonucleotide which has been obtained is desalinated by filtering
it through an NAP-5.RTM. column (PHARMACIA-LKB BIOTECHNOLOGY,
UPPSALA, Sweden).
EXAMPLE 3
Preparation of an Active Oligonucleotide in the Form of an
N-Hydroxysuccinimide Ester
The modified oligonucleotide (designated pyr-T.sub.10 -NH2 below),
prepared in accordance with the method described in Example 2
above, is converted into its corresponding activated intermediate
(N-hydroxysuccinimide ester) in accordance with the following
protocol:
The lyophilized oligonucleotide pyr-T.sub.10 -NH2 (10 to 25 nmol)
is taken up in 12 .mu.l of 50 mM N-(3-sulphopropyl)morpholine
(MOPS) buffer, pH 7.0. 28 .mu.l of dimethylformamide containing 4
.mu.mol of disuccinimidyl suberate (DSS, PIERCE ROCKFORD, Ill.) are
added to this solution and the mixture is then left to stir
mechanically (16 hours at 4.degree. C. or 5 hours at 20.degree.
C.). The reaction mixture is loaded onto an NAP-5.RTM. column which
has been equilibrated with water and the column is eluted with
water in accordance with the protocol recommended by the
manufacturer. The excluded fraction (1 ml) is extracted 5 times
with 1 ml of n-butanol. At each extraction, the mixture is
centrifuged, after which the upper (organic) phase is discarded and
the lower (aqueous) phase is retained. After the last extraction,
the N-hydroxysuccinimide ester (designated pyr-T.sub.10 -NHS
below), which has sedimented to the bottom of the tube, is dried in
vacuo (SPEED-VAC) and then stored at -20.degree. C. (preferably for
less than 24 hours) until it is used.
An analytical HPLC on a LICHROSPHER.RTM. RP-18E/125-4 (5 .mu.m)
column (MERCK, DARMSTADT, Germany) is carried out on an aliquot of
the reaction mixture at different reaction times; a gradient of
acetonitrile in 50 mM triethylammonium acetate (buffer A: 5%
acetonitrile, buffer B: 50% acetonitrile) is used for the elution;
flow rate, 1 ml/min, gradient of 10% B to 30% B in 20 minutes, then
of 30% B to 50% B in 15 minutes. This chromatographic analysis
shows the disappearance of the pyr-T.sub.10 -NH.sub.2
oligonucleotide peak (retention time, approximately 19 minutes) and
the appearance of a peak (retention time, approximately 27 minutes)
corresponding to the N-hydroxysuccinimide ester (designated
pyr-T.sub.10 -NHS).
EXAMPLE 4
Coupling a Modified Oligonucleotide to Peptides
A) Coupling to the ACTH(11-24) Fragment
20 nmol of pyr-T.sub.10 -NHS, obtained as described in Example 3,
are taken up in 50 ml of MOPS buffer (50 mM, pH 7.8). The
ACTH(11-24) peptide (SIGMA, St Louis, United States) (26 nmol, 1.3
eq.) is added in 50 .mu.l of MOPS buffer, pH 7.8. The mixture is
incubated at 4.degree. C. overnight.
The disappearance of pyr-T.sub.10 -NHS and the appearance of a
major peak corresponding to the oligonucleotide-peptide conjugate
(retention time, approximately 26.4 minutes) are seen in an
analytical HPLC which is carried out under conditions which are
identical to those of Example 3.
The reaction mixture is purified by semi-preparative HPLC carried
out on a LICROSPHER.RTM. RP-18E/125-4 (5 .mu.m) column using a
gradient of acetonitrile in 50 mM triethylammonium acetate. The
elution is carried out under the same conditions as the analytical
HPLC described in Example 3.
The oligonucleotide-peptide conjugate is detected, by measuring the
UV absorbance at 265 nm, and collected in the fraction
corresponding to a retention time of between 24 and 25.5 minutes;
this fraction is dried by evaporation (SPEED-VAC). This results in
approximately 3.7 nmol of conjugate designated pyr-T.sub.10
-ACTH(11-24).
B) Coupling to the ACTH(18-39) Fragment
The oligonucleotide ester pyr-T.sub.10 -NHS (EX.3) (approximately
20 nmol) is coupled to approximately 35 nmol, that is approximately
1.8 eq., of the ACTH(18-39) fragment (SIGMA, St Louis, United
States), and the coupling product, designated pyr-T.sub.10
-ACTH(18-39) is purified using the protocols described in A)
above.
The pyr-T.sub.10 -ACTH(18-39) conjugate is detected in the fraction
corresponding to a retention time of between 26 and 27 minutes.
After this fraction has been dried, approximately 7 nmol of the
conjugate are obtained.
EXAMPLE 5
Synthesis of a Pyrrole-T.sub.10 -Biotin Conjugate
A modified oligonucleotide having the sequence pyr-T.sub.10 -NH2
(from 10 to 20 .mu.mol) is treated with an excess of biotin-NHS
(approximately 50 equivalents) (SIGMA) dissolved in 20 .mu.l of
dimethylformamide in a carbonate buffer (1 M) at pH 9; the mixture
is incubated at 20.degree. C. for 30 minutes.
The reaction mixture is purified on an exclusion column (NAP
PHARMACIA) and the pyr-T.sub.10 -bio conjugate (Rt=23 minutes) is
separated from the residual pyr-T.sub.10 -NH2 oligonucleotide
(Rt=18 minutes) by means of analytical HPLC runs carried out under
the conditions of Example 3. The fractions are detected at 265
nm.
EXAMPLE 6
Electropolymerization of the Pyr-T.sub.10 -Peptide and Pyr-T.sub.10
-Biotin Conjugates
The conjugates, which have been synthesized as described in
Examples 4A), 4B) and 5, are deposited, by means of
electropolymerization, on gold-covered silicon microelectrodes.
These microelectrodes are arranged so as to create a matrix in
"draught-board" form; this matrix is formed from square
microelectrodes with 50 .mu.m sides arranged in 5 columns and 4
rows in accordance with the following scheme (n=5 columns and p=4
rows). The microelectrodes are identified by their co-ordinates
(i;j).
The electropolymerization is carried out by immersing the electrode
matrix in a medium containing the pyrrole-oligonucleotide conjugate
biomolecule concerned, and pyrrole (molar ratio of
pyrrole/conjugate=approximately 10,000) in an 0.1 M solution of
LiClO.sub.4. The electrode onto which it is desired to effect the
deposition is connected to a potentiostat, and cycles of between
-0.35 V and +0.85 V (potentials measured in relation to a calomel
electrode connected to the electrolytic cell and connected to the
potentiostat) are carried out at a rate of 100 mV/s. The
counterelectrode consists of a platinum wire.
Some of the microelectrodes are covered with a copolymer formed
from polypyrrole and a conjugate:
pyr-T.sub.10 -ACTH (18-39): electrodes (1;4) and (3;4)
pyr-T.sub.10 -bio: electrodes (2;3) and (4;4).
The remaining electrodes are either left in their initial state,
ie. the gold deposit remains unchanged (GOLD), or else covered with
unmodified polypyrrole (PP); these electrodes constitute negative
controls which make it possible to assess the specificity of the
reactions carried out on the matrix.
Table I below shows the microelectrode matrix, and the location of
the different deposits, in diagram form.
TABLE I ______________________________________ i 1 2 3 4 5
______________________________________ 1 GOLD GOLD GOLD ACTH GOLD 2
GOLD GOLD BIO GOLD GOLD 3 PP PP PP ACTH PP 4 PP PP PP BIO PP
______________________________________ GOLD: no deposit PP:
polypyrrole deposit
EXAMPLE 7
Demonstration of Electropolymerized Biomolecules on the
Electrodes
The electrode matrix constructed in Example 6 is incubated in the
presence of a streptavidin-phycoerythrin conjugate (1 mg/ml
MOLECULAR PROBES commercial solution of
streptavidin-R-phycoerythrin diluted 1/20 in 10 mM phosphate
buffer, pH 7.4, containing 0.5M NaCl and 0.05% TWEEN 20).
Observation under an epifluorescence microscope reveals that the
fluorescence is located on electrodes (2;3) and (4;4). There is no
interfering fluorescence on the other electrodes, demonstrating
that the pyrrole-oligonucleotide-biotin conjugate attached
specifically to the desired target electrodes.
The same microelectrode matrix is incubated in the presence of a
biotinylated antibody which is specific for the C-terminal moiety
of the ACTH 18-39 peptide (ACR-17-bio antibody: CIS BIO
INTERNATIONAL) and then in the presence of a
streptavidin/phycoerythrin conjugate in order to reveal the product
of the reaction.
Observation under an epifluorescence microscope reveals that only
the microelectrodes carrying the pyrrole-oligonucleotide-ACTH
conjugate and those carrying biotin are fluorescent, demonstrating
that the pyrrole-oligonucleotide-ACTH conjugate attached
specifically to the desired target electrodes, that it is
accessible to an antibody and that its antigenic properties have
been retained.
EXAMPLE 8
Using the method described in Example 4, the ACTH(18-39) fragment
is coupled to an oligonucleotide-pyrrole ester of
N-hydroxysuccinimide which has been prepared, using the method
described in Example 3, from an oligonucleotide-pyrrole which has
the sequence 5'pyr-T.sub.9 -NH2 and which was synthesized in
accordance with the protocol described in Example 2.
The following conjugates of the P-O-M type are thus available:
______________________________________ I. pyr-T.sub.10 -ACTH(11-24)
II. pyr-T.sub.10 -ACTH(18-39) III. pyr-T.sub.9 -ACTH(18-39) IV.
pyr-T.sub.10 -biotin V. pyr-T.sub.5 -HCVG(formed by combining a
T.sub.5 arm and a specific sequence of the genome of the HCV
virus). ______________________________________
Following the electropolymerization method described in Example 6,
conjugates I to V are electrochemically deposited on a lattice of
square microlectrodes having sides of 100 .mu.m.
Table II below depicts the microelectrode lattice, and the location
of the different deposits, in diagram form.
Some electrodes are left uncovered (GOLD) or covered with
unmodified polypyrrole (PP).
TABLE II ______________________________________ i 1 2 3 4 5 6 7 8
______________________________________ 1 GOLD IV I I III GOLD V PP
2 PP III II II IV GOLD V GOLD
______________________________________
This microelectrode plate is incubated with an oligonucleotide
probe which has the sequence dC.sup.biotin (dA).sub.9 and which was
prepared and purified in accordance with Example 2 using the
phosphoramidite biotin described by ROGET et al. [Nucleic Acids
Res., 17, 7643-7650 (1989)].
The hybridization is carried out, at 20.degree. C. for 30 min and
then at 4.degree. C. for 30 min, using approximately 8 pmol of
oligonucleotide probe in 200 .mu.l of hybridization buffer (0.1M
phosphate buffer, pH 7.4, containing 0.5 M NaCl/TWEEN.
Visualization is then effected by incubating (at 4.degree. C. for
10 min) in a solution of streptavidin-phycoerythrin (1 mg/ml
MOLECULAR PROBES commercial solution of
streptavidin-R-phycoerythrin) which has been diluted 1/20 in
hybridization buffer.
After examination under a fluorescence microscope, it is observed
that, while electrodes (1;2) and (2;5) are strongly positive and
electrodes (1;3), (1;4), (2;3) and (2;4) are positive, electrodes
(1;5) and (2;2) exhibit a weaker fluorescence; all the other
electrodes, including (1;7) and (2;7), do not exhibit any
fluorescence above the background noise.
Conjugate IV (pyr-T.sub.10 -biotin) serves as a positive
visualization control. It guarantees visualization by the
streptavidin/phycoerythrin complex when the plate is used for the
first time. The 4 microelectrodes carrying the P-O-M-type conjugate
in which O is an oligonucleotide having the T.sub.10 sequence, i.e.
(1;3), (1;4), (2;3) and (2;4), exhibit a positive hybridization,
thereby guaranteeing that the P-O-M-type compound was copolymerized
on these electrodes.
The weak hybridization observed on electrodes (1;5) and (2;2), and
the absence of hybridization on electrodes (1;7) and (2;7),
demonstrate the selectivity of the hybridization and illustrate the
lower limit for the size of the oligonucleotide moiety of the
conjugate which permits this dilution [sic] by hybridization to be
effected and which may be estimated to be a length of between 5 and
9 nucleotides.
EXAMPLE 9
The P-O-M-type conjugate Pyr-T.sub.10 -Bio described in Example 5
is used in solution in water at a concentration of 140 A.sub.265
U/ml, enabling the molar concentration, that is 1.17 .mu.mol/ml
(where .epsilon..sub.265 =120,000) to be determined due to the
UV-absorbing properties of the O moiety of this conjugate.
A range of consecutive dilutions of the conjugate (1/5; 1/25; 1/50;
1/250; 1/1250) is prepared in water, and 10 .mu.l of each dilution
is added to 300 .mu.l of a 20 mM solution of pyrrole in 0.1M
LiClO.sub.4.
Copolymerizations are carried out on microelectrodes, as described
in Example 6, using solutions containing varying concentrations of
the pyr-T.sub.10 -Bio conjugate. The pyrrole/conjugate molar ratio
is different for each electrode.
Table III below shows the microelectrode lattice and the location
of different deposits, in diagram form.
TABLE III ______________________________________ i 1 2 3 4 5 6
______________________________________ 1 GOLD 1/5 1/25 1/50 1/250
1/1250 2 PP 1/5 1/25 1/50 1/250 1/1250
______________________________________
Visualization is effected by incubating a 1/20 solution of
streptavidin/phycoerythrin in a 10 mM phosphate buffer, pH 7.4,
containing 0.5M NaCl and 0.05% Tween 20.
The electrode plate is observed under an epifluorescence microscope
which is coupled to a CCD (HAMAMATSU) camera which is itself
connected to a microcomputer equipped with an image analysis
program.
The time of exposure can be arranged in order to vary the
sensitivity of the detection.
Using exposure times of 0.5 s, 1 s, 2 s, 4 s, 8 s and 16 s, it is
observed that at least the three contiguous contacts (1;2), (1;3)
and (1;4) always give fluorescent signals which are of increasing
intensity (1;2>(1;3)>1;4). Contacts (1;1) and (2;1 [sic])
give the background noise value).
The signals supplied by each member of the contact pairs (1;2) and
(2;2), (1;3) and (2;3), etc. are of the same order, giving an
indication of the reproducibility.
This demonstrates that the fluorescence intensities are correlated
with the initial quantities of P-O-M-type compound (originally
quantified by absorption at 265 nm) introduced into the different
electrolyte solutions.
EXAMPLE 10
a) Variable quantities of a pyrrole-oligonucleotide conjugate
(pyrrole-T.sub.10 -k-ras), having the formula
Pyr-5'(T.sub.10)-k-ras.sub.(28-41), in which (28-41) represents the
sequence of nucleotides 28 to 41 of the sense strand of the human
k-ras sequence carrying a G.fwdarw.A mutation at nucleotide 34,
were electropolymerized, as described in Example 6, in the presence
of a constant quantity of pyrrole (600 .mu.l of 20 mM pyrrole in
LiClO.sub.4, that is 1.2.times.10.sup.-5 mol), onto 5 different
microelectrodes of a plate of gold-covered square microelectrodes
(50 .mu.m.times.50 .mu.m), as indicated in Table IV below.
TABLE IV ______________________________________ Deposit Quantity of
conjugate Pyrrole/conjugate ratio
______________________________________ 1 3 .times. 10.sup.-9 mol 4
000 2 6 .times. 10.sup.-10 mol 20 000 3 1.2 .times. 10.sup.-10 mol
10.sup.5 4 2.4 .times. 10.sup.-11 mol 5 .times. 10.sup.5 5 4.3
.times. 10.sup.-12 mol 2.5 .times. 10.sup.6
______________________________________
b) The same procedure is carried out on 5 other microelectrodes of
the same plate using the P-O-M-type conjugate pyrrole-T.sub.10
-biotin and employing the range shown in Table V below and in FIG.
1:
TABLE V ______________________________________ Deposit Quantity of
conjugate Pyrrole/conjugate ratio
______________________________________ 1 6.6 .times. 10.sup.-9
mol.sup. 1800 2 6.6 .times. 10.sup.-10 mol 18000 3 1.3 .times.
10.sup.-10 mol 9 .times. 10.sup.4 4 2.6 .times. 10.sup.-11 mol 4.5
.times. 10.sup.5 5 5.3 .times. 10.sup.-12 mol 2.25 .times. 10.sup.6
______________________________________
The plate is incubated, at 45.degree. C. for 30 min, in the
presence of the 5'-biotinylated oligonucleotide which is
complementary to the k-ras.sub.(28-41) sequence. A 2A.sub.260 U/ml
solution of biotinylated oligonucleotide is used, with this
solution being diluted 1/1000 in 10 mM PBS buffer, pH 7.4,
containing 0.5M NaCl and 10 mM EDTA. After washing (20.degree. C.)
with 10 mM PBS buffer containing 0.5M NaCl and 0.05% TWEEN 20, the
plate is incubated (at 20.degree. C. for 10 min) in a solution of
streptavidin-phycoerythrin (1 mg/ml MOLECULAR PROBES commercial
solution of streptavidin-R-phycoerythrin) diluted 1/20 in 10 mM PBS
buffer, pH 7.4, containing 0.5M NaCl and 0.05% TWEEN 20.
The plate is observed under an epifluorescence microscope (OLYMPUS)
which is equipped with a CCD camera (integration time t.sub.i =2s;
gain=.times.4) linked to a computer which is equipped with image
analysis software (IMAGE PRO).
The fluorescence measurements obtained with the ranges of the
pyrrole-T.sub.10 -k-ras and pyrrole-T.sub.10 -biotin conjugates are
shown in FIG. 1: (----=k-ras; --.cndot.--=biotin),
which depicts the fluorescence on each electrode plotted against
the initial quantity of conjugate present in the cell during the
electropolymerization and therefore the pyrrole/conjugate molar
ratio.
This experiment demonstrates that choosing the pyrrole/P-O-M or
pyrrole/oligonucleotide molar ratio makes it possible to control
the quantity of molecules grafted to each electrode.
EXAMPLE 11
A Pyr-T.sub.10 -biotin conjugate is deposited, by
electropolymerization and in accordance with the protocol described
in Example 6, on a microelectrode matrix formed from 50 .mu.m
square electrodes (see Example 6), and following the arrangement
(represented by the i/j coordinates) indicated on the first 2 lines
of Table VI below. 2 series of identical depositions, B.sub.n and
B.sub.n ', are effected; depositions B.sub.1 and B.sub.1 ' are
carried out consecutively using two solutions prepared from the
same solution of conjugate; the electrode and the cell are rinsed
between each deposition. The same procedure is adopted with B.sub.2
and B.sub.2 ', etc. Each series of depositions is carried out using
5-fold dilutions (in water), such as [B.sub.2 ]=[B.sub.1 ]/5;
[B.sub.3 ]=[B.sub.2 ]/5. These dilutions are obtained from a parent
solution of Pyr-T.sub.10 -biotin whose concentration is estimated
to be 125 mmol/l (OD.sub.265 measurement, taking .epsilon..sub.265
=120,000). 8 .mu.l of each dilution is used in 600 .mu.l of
electropolymerization solution (20 mM pyrrole in 0.1M LiClO.sub.4).
The concentrations of Pyr-T.sub.10 -biotin conjugate (C), the
[Pyrrole]/[Pyr-T.sub.10 -biotin] ratios (R), and the quantities of
Pyr-T.sub.10 -biotin conjugate which are initially present in the
electropolymerization cell, are shown in Table VI below for each
B.sub.1 /B.sub.1 '; B.sub.2 /B.sub.2 '; etc. group.
TABLE VI
__________________________________________________________________________
i 1 2 3 4 5 6 7
__________________________________________________________________________
1 GOLD B.sub.1 B.sub.2 B.sub.3 B.sub.4 B.sub.5 GOLD 2 GOLD B.sub.1
' B.sub.2 ' B.sub.3 ' B.sub.4 ' B.sub.5 ' GOLD C(.mu.mol/l) -- 125
25 5 1 0.2 -- R -- 1.2 .times. 10.sup.4 6 .times. 10.sup.4 3
.times. 10.sup.5 1.5 .times. 10.sup.6 7.5 .times. 10.sup.6 --
Q(mol) -- 1 .times. 10.sup.-9 2 .times. 10.sup.-10 4 .times.
10.sup.-1 1 8 .times. 10.sup.-12 1.6 .times. 10.sup.-12 --
__________________________________________________________________________
After washing the electrode plate, visualization is effected by
incubating in the presence of a streptavidin-phycoerythrin
conjugate (at 20.degree. C. for 10 min, commercial solution diluted
1/10 in 10 mM PBS buffer, pH 7.4, containing 0.5M NaCl and 0.05%
TWEEN 20).
After rinsing with the same buffer, the plate is observed under a
fluorescence microscope which is equipped with a CCD HAMAMATSU
camera and IMAGE PRO image analysis software (integration time
t.sub.i =2 s; gain.times.4). Using this software, it is possible to
measure the fluorescence of each deposit point by point.
The results are shown in Table VII below, where the fluorescence is
shown for each deposit in arbitrary fluorescence units (AFU) in
accordance with the coordinate system (i;j) of Table VI, and in
FIG. 2, which depicts the fluorescence in AFU plotted against the
initial quantity of Pyr-T.sub.10 -biotin conjugate in the
electropolymerization cell (=B series; .cndot..sup.- =B'
series).
TABLE VII ______________________________________ i 1 2 3 4 5 6 7
______________________________________ 1 10 206 97 55 21 21 11 2 11
208 100 51 22 12 9 ______________________________________
These results clearly demonstrate a relationship of direct
proportionality between the response (fluorescence) and the initial
quantity of pyrrole-T.sub.10 -biotin conjugate (or the initial
[pyrrole]/[pyr-T.sub.10 -biotin] ratio since the concentration of
pyrrole is constant) in the electropolymerization mixture.
The use of compound [sic] of the P-O-M type therefore makes it
possible to monitor the quantity of pyrrole (pyr-T.sub.10 -bio)
conjugate which is present in an electrolyte solution in a simple
manner and to prepare calibration ranges for electrode
matrices.
* * * * *